Original article Responses to elevated atmospheric CO concentration and nitrogen supply of Quercus ilex L seedlings from a coppice stand growing at a natural CO spring Roberto School of Forest Resources and Conservation, Tognetti* Jon D Johnson* University of Florida, 326 Newins-Ziegler Hall, Gainesville, FL, 2611, USA (Received 15September 1998; accepted March 1999) Abstract - Quercus ilex acorns were collected from a population of trees with a lifetime exposure to elevated atmospheric CO con2 centration (CO and after germination seedlings were exposed at two [CO (370 or 520 μmol mol in combination with two soil ) -1 ), ] N treatments (20 and 90 μmol mol total N) in open-top chambers for months Increasing [CO stimulated photosynthesis and leaf -1 ] dark respiration regardless of N treatment The increase in photosynthesis and leaf dark respiration was associated with a moderate reduction in stomatal conductance, resulting in enhanced instantaneous transpiration efficiency in leaves of seedlings in CO enriched air Elevated [CO increased biomass production only in the high-N treatment Fine root/foliage mass ratio decreased with ] high-N treatment and increased with CO enrichment There was evidence of a preferential shift of biomass to below-ground tissue at a low level of nutrient addition Specific leaf area (SLA) and leaf area ratio (LAR) decreased significantly in leaves of seedlings grown in elevated [CO irrespective of N treatment Leaf N concentration decreased significantly in elevated [CO irrespective of N 2] ] treatment As a result of patterns of N and carbon concentrations, C/N ratio generally increased with elevated [CO treatment and ] decreased with high nutrient supply Afternoon starch concentrations in leaves did not increase significantly with increasing [CO ], as was the case for morning starch concentrations at low-N supply Starch concentrations in leaves, stem and roots increased with elevated [CO and decreased with nutrient addition The concentration of sugars was not significantly affected by either CO or N ] 2 treatments Total foliar phenolic concentrations decreased in seedlings grown in elevated [CO irrespective of N treatment, while ] nutrient supply had less of an effect We conclude that available soil N will be a major controlling resource for the establishment and growth of Q ilex in rising [CO conditions © 1999 Éditions scientifiques et médicales Elsevier SAS ] carbon physiology / elevated [CO / natural CO springs / nitrogen / Quercus ilex ] 2 Résumé - Réponses de jeunes plants de Quercus ilex L issus d’une population poussant dans une zone naturellement enrichie en CO une concentration élevée de CO dans l’air et un apport d’azote Des jeunes plants de Quercus ilex L., issus d’une , 2 population d’arbres ayant poussé dans une concentration élevée de CO ont été exposés deux concentrations en CO , 2 -1 -1 (370 μmol mol ou 520 μmol mol en combinaison avec deux fertilisations du sol en azote (20 et 90 μmol mol N total) dans des ) -1 chambres ciel ouvert pendant six mois L’augmentation de concentration en CO stimule la photosynthèse et la respiration nocturne des feuilles indépendamment du traitement en azote Les augmentations de photosynthèse et de la respiration nocturne des feuilles ont été associées une réduction modérée de conductance stomatique, ayant pour résultat d’augmenter l’efficience transpiratoire instantanée des feuilles des jeunes plants cultivés en CO élevé L’augmentation de concentration du CO accrt la production de 2 biomasse seulement dans le traitement élevé en azote Le rapport des racines fines la masse de feuillage a diminué avec le traite- * Correspondence and reprints: Istituto per l’Agrometeorologia e l’Analisi Ambientale applicata all’Agricoltura, Consiglio Nazionale delle Ricerche, via Caproni 8, Firenze, 50145, Italy ** Present address: Intensive Forestry Program, Washington State University, 7612 Pioneer Way E., Puyallup, WA-98371-4998, USA tognetti@sunserver.iata.fi.cnr.it .La surface spécifique de feuille (SLA) et les taux de la surface de feuille ment en azote et a augmenté avec l’enrichissement en (LAR) ont diminé de manière significative pour les feuilles des jeunes plants développés sous une concentration élevée de indépendamment du traitement en azote La concentration en azote des feuilles a diminué de manière significative dans le traitement du traitement en azote En raison des configurations des concentrations d’azote et de carbone, le taux élevé en CO , CO , COindépendamment C/N a augmenté avec le traitement élevé en CO et diminué avec l’apport d’azote Dans l’après-midi, les concentrations en amidon des feuilles n’ont pas augmenté de manière significative avec l’augmentation du CO comme pour les concentrations en amidon dans le , Les concentrations en amidon dans les feuilles, la tige et les racines ont augmenté dans le cas du traitement limité en azote du matin cas du traitement avec une concentration élevée en CO et diminué avec l’apport en azote Les concentrations en sucre n’ont pas été affectées sensiblement par les traitements de CO ou de N Les concentrations phénoliques foliaires totales ont diminué pour les jeunes plants qui ont poussé dans le traitement en CO élevé, indépendamment du traitement en N Nous concluons que la disponibil2 ité en azote dans le sol jouera un rôle majeur dans l’établissement et la croissance de Q ilex dans un environnement caractérisé par un accroissement de la concentration en CO dans l’air © 1999 Éditions scientifiques et médicales Elsevier SAS azote / 2 CO élevé / physiologie du carbone / Quercus ilex / sources naturelles de de CO Introduction in plant N On the other hand, there is efficiency (e.g [4, 31]) increasing evidence that reductions in tissue N concentrations of elevated CO plants is probably a size-grown dependent phenomenon resulting from accelerated plant growth [10, 46] It has also been documented that reductions in plant tissue N concentrations under elevated ] [CO may substantially alter plant-herbivore interactions [30] as well as litter decomposition [13] In fact, insect herbivores consume greater amounts of elevated -grown CO foliage apparently to compensate for their reduced N concentration; again, litter decomposition rates may be slower in elevated [CO environments ] because of the altered balance between N concentrations and fiber contents has been attributed to physiological changes use Atmospheric carbon dioxide concentration [CO is ] -1 currently increasing at a rate of about 1.5 μmol mol annually [52], as a result of increasing fossil fuel consumption and deforestation Moreover, models of future global change are in general agreement predicting levels -1 reaching 600-800 μmol mol by the end of next century -1 from present levels ranging from 340 to 360 μmol mol [12] -enriched CO atmospheres have been shown to increase photosynthetic carbon gain, the growth of plants and concentrations of total non-structural carbohydrates, although there is evidence of species-specific responses (see reviews [1, 2, 7, 16, 42]) The impact of increased ] [CO on plant growth is modified by the nutrient level: growth enhancement in elevated [CO has often been ] shown to decline under nutrient stress Indeed, enhanced growth may increase plant nutrient requirement, but many Mediterranean sites are considered to have low nitrogen (N) availability On the other hand, it has been proposed that plants adjust physiologically to low nutrient availability by reducing growth rate and showing a high concentration of secondary metabolites [5] The carbon-nutrient balance hypothesis predicts that the availability of excess carbon at a certain nutrient level leads to the increased production of carbon-based secondary metabolites and their precursors [39] For instance, the often observed increase in C/N ratio under elevated [CO has led some authors to suggest that ] ] [CO increases might produce changes in the concentration of carbon-based secondary compounds [29], thus affecting plant-herbivore interactions Changes in N availability may also alter per se the concentrations of carbon-based secondary chemicals [18] A major effect of CO atmospheres is the -enriched reduction in the N concentration of plant tissues, which Quercus ilex L is the keystone species in the Mediterranean environment Q ilex forests, once dominant, have shrunk as a result of fires and exploitation for firewood and timber over thousands of years The ability of Q ilex to compete at the ecosystem level as [CO ] continues to increase is of concern While many studies have looked at seedling response to elevated [CO ], nothing is known of progeny of trees growing for long term in a CO atmosphere Extrapolation from -enriched studies on seedlings growing in elevated [CO to mature ] trees should be made only with extreme caution However, the seedling stage represents a time characterized by high genetic diversity, great competitive selection and high growth rates [7] and as such may represent one of the most crucial periods in the course of tree establishment and forest regeneration Indeed, a small increase in relative growth at the early stage of development may result in a large difference in size of individuals in the successive years, thus determining forest com- munity structure [3] As the increase in plant productivity in response to rising [CO is largely dictated by photosynthesis, respi] ration, carbohydrate production and the subsequent incorporation of the latter into biomass [24], the objectives of this study were i) to investigate how CO avail2 ability alters whole-plant tissue N concentration, ii) to examine the effects of increased [CO on carbon alloca] tion to the production of biomass, total phenolic compounds and TNC (total non-structural carbohydrates, starch plus sugars), and finally iii) to determine how elevated [CO influences gas exchange rate in progeny of ] Q ilex trees growing in a CO environment -enriched under two different levels of N The parent trees grow in poor soil nutrient conditions under long-term CO enrichment and their carbon physiology has been the object of a previous study [48] We hypothesized that the juvenile stage would behave like acclimated parent trees when grown in similarly poor soil nutrient conditions Materials and methods 2.1 Plant material and growth conditions Acorns of Q ilex were collected in December from adult (open-pollinated) trees, growing in the proximity of the natural CO spring of Bossoleto and which have spent their entire lifetime under elevated [CO the CO ]; vent is located in the vicinity of Rapolano Terme near Siena (Italy) (for details see [28]) Seeds were immediately sent to USA and sown in PVC pipe tubes (25 cm ) height x 5.5 cm averaged internal diameter, 600 cm After germination, seedlings were thinned to one per pot The tubes were filled with a mixture (v/v) of 90 % sand and 10 % peat, a layer of stones was placed at the base of each tube The first stage of growth was supported by adding commercial slow-release Osmocote (18:18:18, N/P/K); the nutrient additions were given in one pulse of g, applied after month of growth in the tubes Soil nutrients in terrestrial systems suggest that N mineralization is sometimes limited to short periods early in growing season; furthermore, by giving an initial pulse of nutrients, we created a situation in which plant requirements for nutrients were increasing (due to growth) while supply was decreasing (due to uptake) [10], a phenomenon that may occur particularly in natural systems low in soil N During the first month of growth (January 1995) the seedlings were fumigated twice with a commercial fungicide Two hundred and forty seedlings were grown for months in six open-top chambers located at the School of Forest Resources and Conservation, University of Florida, Austin Cary Forest, approximately 10 km northeast of Gainesville Each chamber received one of two -1 ] 2 CO treatments: ambient [CO or 150 μmol mol exceeding ambient [CO The chambers were 4.3 m tall ] and 4.6 m in diameter, covered with clear polyvinylchlo- ride film and fitted with rain-exclusion tops Details of the chamber characteristics may be found in [23] The , CO supplied in liquid form that vaporized along the copper supply tubes, was delivered through metering valves to the fanboxes of three chambers The COtreat2 ment was applied during the 12 h (daytime) the fans were running with delivery being controlled by a solenoid valve connected to a timer The CO was delivered for about 15 after the fans were turned off in the evenings in order to maintain higher concentrations in the chambers The [CO was measured continuously in ] both the ambient and elevated [CO chambers using a ] manifold system in conjunction with a bank of solenoid valves that would step through the six chamber sample lines every 18 Overall mean daily [CO for the ] -1 above treatments was 370 or 520 μmol mol at present or elevated [CO respectively (for details see [26]) The ], 2 ] [COduring the night remained higher in the CO enriched chambers, since the fans were turned off, avoiding air mixing beginning of March (1995), two different nutrisolution treatments were initiated Within a chamber, equal numbers of pots (21) were randomly assigned to a high- or low-N treatment Before starting the nutrient treatment, the superficial layer of Osmocote was removed from the tubes and the latter flushed repeatedly for week with deionized water in order to remove accumulated salts and nutrients The seedling containers were assembled in racks and wrapped in aluminum foil to avoid root system heating, and set in trays constantly containing a layer of nutrient solution to avoid desiccation and minimize nutrient loss limiting nutrient disequilibrium [25] At the ent Plants were fertilized every days to saturation with of the two nutrient solutions obtained by modifying a water soluble Peters fertilizer (HYDRO-SOL®, GraceSierra Co., Yosemite Drive Milpitas, CA, USA): com-1 plete nutrient solution containing high N (90 μmol mol ), NO NH or a nutrient solution with low N (20 μmol -1 mol NH Both nutrient solutions contained PO ) NO ), -1 ), -1 (20.6 μmol mol K (42.2 μmol mol Ca (37.8 μmol ), -1 ), -1 ), -1 mol Mg (6 μmol mol SO (23.5 μmol mol Fe ), -1 ), -1 (0.6 μmol mol Mn (0.1 μmol mol Zn (0.03 μmol ) -1 ), -1 ), -1 mol Cu (0.03 μmol mol B (0.1 μmol mol and Mo (0.02 μmol mol and were adjusted to pH 5.5 ), -1 Every weeks supplementary Peters (S.T.E.M.) micronutrient elements (0.05 g dm were added ) Deionized water was added to saturation every other day in order to prevent salt accumulation Plant containers were moved frequently in the chambers in order to avoid one positional effects 2.2 Gas exchange measurement Measurements of stomatal conductance (g and leaf ) s carbon exchange rate were made with a portable gas analysis system (LI-6200, Li-cor Inc., Lincoln, NE, USA) on upper-canopy fully expanded leaves of the same stage of development of randomly selected 18 plants for each CO x N treatment combination (all mea2 surements were made in duplicate and each leaf was measured twice) Measurements of daytime g and phos tosynthetic rate (A) were performed under saturating -2 -1 ), light conditions (PAR 000-1 500 μmol m s between 10:00 to 15:00 hours on August 27-29 (air temperature 27-30 °C, relative humidity 70-75 %) Leaf dark respiration (R was measured before sunrise ) d (04:00-06:00 hours) on August 26-28 (air temperature, 23-25 °C) Instantaneous transpiration efficiency (ITE) was calculated as A/g Air temperature, relative humidi s ty and PPFD in the leaf cuvette were kept at growth conditions 2.3 Biomass allocation Heights and root-collar diameters were measured on plants (240) on September On September all plants were harvested and were separated into leaves, all the stem, and coarse (> mm) and fine (< mm) roots Surface area of each leaf and total foliage area of each seedling were measured with an area meter (Delta-T Devices Ltd, Cambridge, UK) Plant material was dried at 65 °C to constant weight and dry mass (DW) measurements were made Leaf area ratio, LAR (m g was -1 ), calculated as the ratio of total leaf area to total plant dry mass; specific leaf area, SLA (m g as the ratio of -1 ), total leaf area to leaf dry mass; partitioning of total plant ), -1 dry mass, LWR, SWR and RWR (g g as the fraction of plant dry mass belonging to leaves, stem and roots, respectively In addition, root/shoot dry mass ratio, RSR (g g was determined ), -1 2.4 Carbohydrate, carbon and N analysis The amount of total non-structural carbohydrates measured using the anthrone method on 12 seedlings for each CO x N treatment combination These seedlings were harvested either at dawn or in late afternoon, and immediately (after leaf area measurements) placed into a drier (see above) Previously dried plant materials (leaves, stem and roots) were ground in a Wiley mill fitted with 20 mesh screen Approximately 100 mg of ground tissue was extracted three times in boiling 80 % ethanol, cen- (TNC), including starch and sugars, was trifuged and the supernatant pooled The pellet was digested at 40 °C for h with amyloglucosidase from Rhizopus (Sigma Chemical Co., USA) and filtered Soluble sugars and the glucose released from starch were quantified spectrophotometrically following the reaction with anthrone All samples were prepared in duplicate Total carbon and N concentrations (mg gDW) were -1 determined for all 240 seedlings (leaves, stem and roots) by catharometric measurements using an elemental analyser (CHNS 2500, Carlo Erba, Milano, Italy) on 5-9 mg of powder of dried samples 2.5 Phenolic analysis Equal-aged leaves (three leaves per plant) were taken from all 240 seedlings, the day before the harvest, for total phenolic compounds analysis Leaves were put into liquid N at the field site, then transported to the laboratory and stored in the freezer at -20 °C until analysis The leaf blades were punched on either side of the main vein Five punches (0.2 cm each) per leaf were analyzed for phenolics by modifying the insoluble polymer bonding procedure of Walter and Purcell [51] Other punches from the remaining leaf blades were used for dry mass determination, as described above Leaf tissue was homogenized in 5.0 cm of hot 95 % ethanol, blending and boiling for 1-2 Homogenates were cooled to room temperature and centrifuged at 12 000 g for 30 at 28 °C Supernatants were decanted and evaporated to ) dryness in N at 28 (C Aliquots (8 cm of the sample in 0.1M phosphate buffer (KH pH 6.5) were mixed , PO with 0.2 g of Dowex resin (Sigma Chemical Co., St Louis, MO, USA) by agitating for 30 (200 g, 28 °C) Dowex, a strong basic anion-exchange resin (200-400 dry mesh, medium porosity, chloride ionic form), was purified before use by washing with 0.1 N NaOH solution, distilled water and 0.1N HCL and, finally, with distilled water Absorbance at 323 nm (A was mea) 323 sured spectrophotometrically both before and after the Dowex treatment, representing the absorbance by phenolic compounds Phenolic concentration was determined from a standard curve prepared with a series of chlorogenic acid standards treated similarly to the tissue extracts and comparing changes in absorbance measured for the standards and those caused by the treatment 2.6 Statistical analysis Individual measurements were averaged per plant, and plants measured with respect to each CO x N treat2 ment combination were averaged across the open-top chambers Statistical analyses consisted of two-way of variance (ANOVA) for randomized design and Duncan’s mean separation test for the measured parameters (5 % significant level); CO and N were treated as fixed variables A preliminary analysis showed that differences between chambers within the same CO treatment were never significant Proportions and percentages were transformed using the arcsine of the analysis square root prior to analysis As a result of increases in A and decreases in g ITE , s of leaves increased with [CO in both N supply treat] ments (table I) ITE was significantly different among the four COx N treatment combinations (ITE was high2 er with high-N supply), and there was a significant interaction between COand N treatment (P < 0.05) reflected by a marked increase in ITE in plants grown in elevated ] [CO with a high-N supply The ratio of internal Results 3.1 Gas ]) 2i [CO (C nal) [CO (C decreased (P ]) 2a < to ambient (i.e exter- 0.0001) with both CO enrichment and high-N supply, while the interaction between COand N treatment was not significant exchange rate (table I) The increase in A Increasing [CO had a significant effect on leaf car] bon exchange rate (table I) Comparison of assimilation rates at the growth [CO showed that increasing [CO ] ] -1 from 370 to 520 μmol mol resulted in 33 % increase in A for plants grown with low-N supply and in 36 % increase for plants grown with high-N supply Nutrient supply also significantly affected the response of A Plants grown with high-N supply had 25 and 29 % higher A than plants grown with low-N supply at ambient and elevated [CO respectively There was no strong inter], action between CO and N treatment (P 0.084), i.e increase in [COelicited a similar increase in A in both ] N treatments = Comparison of g at the growth [CO showed that ] s -1 increasing [CO from 370 to 520 μmol mol led to a ] 14 % decrease in g for plants grown with low-N supply s and to 10 % decrease with plants grown with high-N supply (table I) Nutrient supply treatment and the interaction between CO and N treatment did not affect signi2 ficatly g s increase in d R(table was associated with a significant I) Comparison of Rat the growth d ] [CO showed that increasing [CO from 370 to 520 ] -1 μmol mol led to 48 % increase for plants grown with low-N supply and to 36 % for plants grown with high-N supply The increase in N supply and the interaction between COand N treatment had less of the increase in R d 3.2 Growth and biomass an effect on partitioning Basal stem diameter, number of leaves per plant and foliage area were increased by elevated [CO treatment ] only when Q ilex seedlings were grown in the high-N treatment (table II, figure I) Shoot length and individual leaf area were not influenced by [CO treatment but ] increased with N supply After months of COx N treatment combination there were significant increases in the dry mass of roots and coarse roots of seedlings grown in elevated [CO compared to seedlings grown at ] ambient [CO irrespective of N treatment The interac], tion between CO and N treatment was significant for total, stem, fine root and foliage biomass As a result of this, effects of CO air on whole seedling -enriched growth, stem, fine root and foliage biomass were significant only in the high-N treatment (figures and 2) Fine root/foliage mass ratio decreased with N treatment and increased with CO enrichment (table II, figure 2) result of increased allocation to below-ground were increased significantly by CO treatment, while SWR and LWR were decreased, only at a low level of N supply (table II, figure 3) More biomass was partitioned to above-ground tissue in the high-N treatment irrespective of CO treatment; as a result RSR and RWR decreased, while conversely, SWR and LWR increased significantly at a high level of N supply SLA and LAR decreased significantly in leaves of seedlings grown in elevated [CO irrespective of N ] treatment, while N supply affected LAR (only in elevated [CO but not SLA (table II, figure 3) ] As a tissue, RSR and RWR 3.3 Carbon and N concentrations decreased significantly in elevated [CO irrespective of ] nutrient treatment, while N concentrations in stem and roots were decreased by elevated [CO in the high-nutri] ent treatment Nutrient supply treatment affected N concentration significantly in leaves irrespective of CO treatment, and in stem and roots only in the ambient ] [CO treatment As a result of patterns of N and carbon concentrations, C/N ratio generally increased with elevated [CO and decreased with high nutrient supply ] (table III) 3.4 Total non-structural carbohydrate and total phenolic concentrations Morning starch concentrations were higher (P < 0.01) in leaves of seedlings grown in CO air (table -enriched IV), but particularly at low level of N supply Afternoon starch concentrations did not increase significantly with increasing [CO Both morning and afternoon sugars ] concentration did not increase significantly with rising ] [CO Both morning and afternoon starch concentrations decreased (P < 0.001) with increasing N addition while sugars concentration was not affected by N treatment Overall, carbon concentrations in leaves, roots were not significantly nutrient treatment affected by stem either and CO or (table III) Leaf N concentration (table IV) Overall starch concentrations in leaves, stem and roots increased with rising [CO and decreased with N addi] tion (table V) The concentration of sugars was not significantly by either COor N treatment As a were influenced by both CO enrichment and N treatment because of changes in starch affected result, TNC concentrations concentrations concentrations decreased in leaves of in elevated [CO irrespective of N ] treatment, while N supply treatment and the interaction between COand N treatment had less of an effect (fig2 Total phenolic seedlings grown ure 4) Discussion Photosynthesis elevated ] [CO of Q ilex seedlings was stimulated by in the low level of supplemental fer- even tilization and despite declining foliar N concentration, as for other broad-leaved trees (e.g [34]) The increase in leaf dark respiration expressed on a leaf area basis in -enriched CO air may be correlated with the enhanced carbohydrate content [44] Although many studies show significant reductions in plant respiration in elevated ] [CO (e.g [47]; see [1] for a review), accordingly with these seedlings, parent Q ilex trees at the natural CO spring in Italy grow in a N poor soil and have also been found to show higher photosynthesis and dark respiration than trees at ambient [CO [9, 48] Stomatal ] response to COis a common phenomenon and stomatal conductance in many plants decreases in response to increasing [CO (see reviews [1, 7, 42], and references ] cited therein) In our study, however, elevated [CO ] treatments did not strongly alter leaf conductance Similar results have been reported for other species when were grown at high irradiances (e.g [6, 21, 31, 49] Stomatal sensitivity to CO in our seedlings, grown at full irradiances with an adequate supply of soil water, may have been reduced [16] Indeed, the ratio of internal plants ] [CO (demand) to external [CO (supply) decreased ] 2 CO enrichment while intercellular [CO remained ] relatively constant, despite at elevated [CO intercellu] lar [CO should rise if stomata close consistently This ] with implies that as a result of strongly increased assimilation cols) indicate that the interactive effects of CO and nutrient availability are species dependent The lack of a growth response to elevated [CO in seedlings in the ] low-N treatment is of interest because suboptimal concentrations of N are common in the Mediterranean environment Indeed, responses in the parent Q ilex trees at the natural CO springs in Italy not appear to be clear2 ly more evident than in trees at ambient [CO [22] ] and, secondarily decreased stomatal conductance, instantaneous transpiration efficiency of leaves markedly increased at elevated [CO [15] ] rate The elevated [CO treatment increased seedling ] growth only when nutrient availability was high Similar findings have been reported for Pinus taeda L [20], Betula populifolia Marsh., Fraxinus americana L., Acer rubrum L [3] and Pinus palustris Mill [38] However, positive growth responses to CO air even -enriched under conditions of low soil nutrient availability have been reported for Castanea sativa Mill [17], Pinus ponderosa Dougl ex Laws [27], Eucalyptus grandis W Hill ex Maiden [11]and Quercus virginiana Mill [46]; in this latter case the experimental conditions were the same as in the present study These contrasting results (even between studies with identical experimental proto- Coarse root (and total root) biomass responded positively to elevated [CO irrespective of nutrient availabil] ity, while fine root biomass increased significantly under low nutrient availability Partitioning of resources was reflected by adjustments in shoot and root growth and in RSR Low nutrient supply enhanced overall biomass partitioning to roots (higher RWR and RSR, lower SWR), while high-N availability resulted in a greater proportion of biomass being distributed to stem and leaves [19, 32, 38] Preferentially induced distribution of photosynthates below-ground as carbon supply increases in response to -enriched CO air is a common phenomenon [7, 38, 43] Such a pattern was detected in our experiment at a low level of nutrient supply only, which was reflected by increased RSR and RWR This may allow seedlings in -enriched CO air to explore the soil in order to attain such as water and nutrients to meet demands Conversely, seedlings grown in ambigrowth ent [CO had a greater proportion of biomass distributed ] to above-ground tissues at low level of nutrient supply only, which was reflected by decreased RSR and increased SWR and LWR Increased stem biomass in seedlings grown under elevated [CO and high nutrient ] availability was associated with increased stem diameter more resources and height, while in the low soil nutrient availability treatment, seedlings in elevated [CO were even shorter ] than those in ambient [CO ] These findings support the idea that plants allocate photosynthate to tissues needed to acquire the most limiting resources [8] Such shifts to below- and/or aboveground tissues, may have implications during the regeneration phase in terms of competition for light and water with other woody species of the Mediterranean vegetation CO treatment also increased the fine root/foliage mass ratio while N treatment had the opposite effect [46] This change in allocation might represent a substitution between potential carbon assimilation and nutrient acquisition [34] However, conflicting results are report- ed in the literature [37, 45] LAR and LWR decreased in response to elevated [CO suggesting that canopy-level ] adjustment in carbon assimilation did occur in these seedlings It must be pointed out that our seedlings grew in pots and growth responses to elevated [CO may ] sometimes be influenced by pot size, though the issue of pot size is far from being resolved Soil nutrient disequilibrium (which we tried to minimize) may be more important than pot size in affecting growth response to elevated [CO Plants in natural environments not ] have unlimited below-ground resources with which to maximize growth in elevated [CO [1], and the presence ] of shallow bedrock at the site of origin of Q ilex parent trees is, in this sense, a good example The observed increased leaf biomass and area in response to COenrichment (at a high level of soil nutri2 ent availability), as a result of an increase in leaf number rather than leaf size, could affect whole-plant photosynthetic capacity [38] Decreases in SLA have been observed in plants grown in CO air (e.g [14]) -enriched and have been attributed to an additional cell layer [40] or starch accumulation [36] In our study, lower SLA at elevated [CO was partly attributable to higher carbohy] drate concentrations, a large part of which was starch The reduction in N concentration in leaves of seedlings grown at elevated [CO irrespective of the N ], availability in the soil, agrees with other studies on tree species [7, 42] Similarly, mature Q ilex trees grown long term under elevated [CO from which the acorns ], were collected, showed a decreased N concentration in leaves when compared to trees grown at ambient [CO ] [48] We found, however, that N concentration in stem and roots did not decrease in the CO air and -enriched low-N treatment combination The decrease in tissue N concentration may be an indirect and size-dependent effect of elevated [CO [10, 46], alternatively it has ] been proposed that starch accumulation dilutes N and lowers its concentration in the tissues (e.g [53]) Carbon concentration was not strongly affected by either treatment (despite an increase in carbon concentration in the -enriched CO air and low-N treatment combination) The C/N ratio in leaves was enhanced by increasing ] [CO and diminishing nutrient supply; this trend was confirmed in stem and roots but to a lesser extent The increase in the C/N ratio could increase carbon storage and the concentration of carbon-based secondary compounds [30], and nutrient cycling in this as in other Quercus species [50] In the current experiment foliar phenolic concentrations decreased in CO air -enriched A variety of phenolic concentration responses to CO enrichment have been observed (e.g [35]) The parallel increase in TNC at elevated [CO may have caused a ] dilution of phenolics If increased [CO reduces in the ] long-term both the N and phenolic concentrations of leaves the consequences for interactions between Q ilex and insect herbivores may be great Nevertheless, tannin concentrations in leaves of the parent trees were positively affected by elevated CO [48] Enhancement of mineral nutrient supply alone (which caused significant growth stimulation) reduced starch and TNC concentrations in leaves, stem and roots CO enrichment stimulated the accumulation of TNC, by strongly increasing starch formation, starch storage seems to be particularly important in Q ilex The lack of carbon flow to soluble sugars suggests a limitation in the partitioning of carbon to this intermediate [54] Rising concentrations of TNC in the leaves may occur, among other reasons [43], because of reductions in sink activity (e.g as a consequence of limiting resources other than ) CO The accumulation of starch in leaves, particularly in the CO air and low-N treatment combina-enriched tion, however, was not accompanied by the reduction in photosynthetic rate, suggesting that the demand for carbohydrates in these seedlings is high This study, which represents the first one on progenies of trees grown long term in CO air, -enriched reports data similar to those conducted on mature trees at CO spring in Italy in soil with poor N availability [48], in that lack of a growth responses to elevated [CO in seedlings in the low-N treatment occurred ] despite increased rate of net photosynthesis All the parameters studied in the present experiment (except for carbon-based defense compounds) follow the findings on parent trees exposed to CO air when seedlings -enriched grown in elevated [CO and low-N availability are taken ] into account It is possible to hypothesize that the available soil 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September all plants were harvested and were separated into leaves, all the stem, and coarse (> mm) and fine (< mm) roots Surface area of each leaf and total foliage area of each seedling were measured... averaged per plant, and plants measured with respect to each CO x N treat2 ment combination were averaged across the open-top chambers Statistical analyses consisted of two-way of variance (ANOVA)